INTRODUCTION

Animal production is seriously affected by heat stress (HS); the United States pork industry is estimated to lose more than $ 300 million a year; while global losses amount to billions of dollars (^{St-Pierre et al., 2003}). HS -induced economic losses are the result of poor sow performance, reduced and inconsistent growth of fattening pigs, lower carcass quality, and increased veterinary costs (^{St-Pierre et al., 2003}; ^{Renaudeau et al., 2011}). The consequences of HS in the low productive performance of the pig may be related to the negative effects on intestinal integrity (^{Pearce et al., 2013}a; ^{Sanz- Fernandez et al., 2014}).

Mammals in HS redistribute blood to the periphery to maximize radiant heat dissipation, resulting in less blood and nutrient flow in the intestine; compromising the intestinal barrier and increasing its permeability (^{Pearce et al., 2013}b); in addition, HS can antagonize digestibility and anabolic pathways in pigs (^{Mani et al., 2012}; ^{Rakhshandeh et al., 2012}). Because Zn requirements increase during heat stress (^{Lagana et al., 2007}), it has been suggested that Zn supplementation could be used to attenuate the serum Zn decrease during periods of high ambient temperatures (^{Chand et al., 2014}; ^{Li et al., 2015}). Zinc in the form of a divalent metal ion, Zn2+, is nutritionally essential for all living organisms (^{Maret, 2013}); is a trace mineral with proven importance for the function of more than 300 enzymes (^{Chasapis et al., 2012}). Pig diets are generally supplemented with inorganic Zn (ZnSO4 or ZnO), to ensure the required contribution, being the inorganic source of ZnSO4 the one with the highest bioavailability (^{NRC, 2012}); however, under normal physiological conditions and with adequate intake, only 5 to 15% of the Zn from the diet is apparently absorbed (^{McDowell, 2003}). It has been suggested that organic sources of Zn are more bioavailable than inorganic forms, and the bioavailability of organic versus inorganic forms increases in the presence of antagonists, such as Ca, P, phytic acid, and crude fiber (^{Schlegel et al., 2013}; ^{Richards et al., 2015}). ^{Ming-Zhe et al. (2016)}, observed that the biological value of organic zinc from zinc methionine (ZnMet) was 64% higher than that of ZnSO4.

The objective of the present study was to determine the effect of zinc methionine supplementation during the gestation-lactation period and the development-completion period, on the productive performance and during the development-completion period on the morphology of the intestinal epithelium of fattening pigs, raised under a warm or cool environment.

MATERIAL AND METHODS

Location of the study area. The study was carried out in the experimental area for fattening pigs of the Faculty of Veterinary Medicine and Zootechnics-UAS, located in the “La Huerta” pig farm, located in Culiacán municipality, Sinaloa, Mexico. The study consisted of two experiments; Experiment 1 was carried out during the months of July to October (warm period), and Experiment 2 in the months of December to March (cool period). The histological analysis of the small intestine samples (jejunum, duodenum and ileum), taken in the trace, were processed in the Pathology Laboratory of the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Sinaloa, according to the proposed procedure by ^{Prophet et al. (1995)}.

Experimental design. 192 pigs were used (96 in each experiment), with an average age of 79 days and 26.39 ± SD4.97 kg of b.w, to which one of four treatments were assigned in a completely randomized experimental design, with factorial arrangement 2 x 2. In each experiment, the treatments consisted of T1 (Control; n = 24), non-supplemented dams- non-supplemented pigs; T2 (ZnDF; n = 24), non-supplemented dams-pigs supplemented with 100 mg of Zn/kg DM; T3 (ZnGL; n = 24), dams supplemented with 100 mg of Zn/kg DM-non-supplemented pigs and, T4 (ZnGL + ZnDF; n = 24), supplemented dams + supplemented pigs.

The diets used are shown in table 1. The pigs used in the experiment were animals from a previous work, piglets of sows (PIC® Lines) that received or did not receive food added with zinc methionine (100 mg/kg DM) during gestation (80-114 d) and lactation (1-21 d); the diets provided were based on corn-soybean meal (table 2). The organic zinc was provided as zinc methionine (ZnMet) from the Zinpro120 premix (Zinpro® 120; contains 12% Zn and 27.3% methionine; Zinc methionine US Patent No. 4.764.633 and 5.430.164; Mexico release note: B00. 02.08.02.02.0398/11).

Handling of animals. The pigs of each experiment, previously weighed and identified, were housed in 12 pens, each one with a space of 9 m2 (6 x 1.5 m), with a concrete floor, equipped with a hopper-type feeder and integrated nipple drinker; 8 pigs (4 males and 4 females) were housed in each pen. During the test period, which lasted 90 days in each experiment, the pigs had permanent access to free access drinking water and food. The food served in each pen was recorded and at the end of each experiment, the pigs were weighed and sent to the slaughterhouse, where they were slaughtered in the TIF slaughterhouse in accordance with ^{NOM-033-SAG/ZOO-2014}. After slaughter, tissue samples were taken from the small intestine, from the portions of the duodenum, jejunum and ileum, 15 pigs from each experiment, which received or did not receive feed supplemented with 100 mg of zinc methionine/kg of feed during the test period.

Measurements: The food served in each pen was recorded weekly. At the end of each experiment, the pigs in each pen were weighed and with the information on feed consumption and weight gain, the feed conversion was obtained. Pig mortality was in each pen recorded and the mortality rate was determined based on this. In the case of the productive behavior test, the experimental unit was the pen.

At the end of each experiment, the pigs were to the slaughterhouse sent, where samples were taken from each segment of the small intestine (duodenum, jejunum and ileum). Fifteen representative pigs from each experiment (hot and cool season) received or not food supplemented with zinc methionine; without considering whether they were the offspring of sows that received feed or not added with zinc methionine during the gestation and lactation period. The samples were in bottles with 10% buffered formalin preserved until histological analysis.

Determination of THI. The temperature (t °C) and relative humidity (RH,%) data were taken with a thermo-hygrometer, located inside the experimental unit, and were recorded daily during the experimental period (tables 3 and 4). The temperature and humidity index (THI) was calculated using the formula THI = [0.8 x room temperature] + [(% RH/100) x (room temperature -14.4)] + 46.4 (^{Mader et al., 2006}).

Intestinal histology. The samples were sent to the Pathology Laboratory of the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Sinaloa, for processing using the routine paraffin embedding technique and subsequent staining with the hematoxylin and eosin technique. For the histological evaluation, a Motic® model BA 201 brand digital binocular microscope was used, equipped with a camera and software to photo-document images. Images of 30 fields were taken from each intestinal section, the length (in microns) of 10 villi was measured; as well as the depth (in microns) of the crypts, and based on this, the average height of the villi of each intestinal section was calculated; as well as the depth of the crypt. Based on this information, the villus height, crypt depth (V:C) ratio was determined; which was used for the corresponding statistical analysis. It is worth mentioning that the samples from each section of the small intestine that did not meet the requirements for histological analysis were discarded and were not considered in the statistical analysis.

Statistical analysis. To the data obtained from daily feed consumption (DFC), daily weight gain (DWG), feed conversion (FC), total weight gain per stage (TWG) and mortality during the experiment; as well as, to the relation villi, crypt. An analysis of variance was applied to them for a completely randomized design, with factorial arrangement (^{Steel and Torrie, 1985}); setting a maximum alpha of 0.05 to accept statistical difference.

𝑌_𝑖𝑗𝑘 = 𝜇 + 𝑍??_𝑖 + 𝐴_𝑗 + 𝑍𝑛𝐴_𝑖𝑗 + 𝐸_{𝑖𝑗𝑘;}

For the analysis of productive performance, the mathematical model was the following:

𝑌_𝑖𝑗𝑘 = 𝜇 + 𝑍𝑛_𝑖 + 𝐴_𝑗 + 𝑍𝑛𝐴_𝑖𝑗 + 𝐸_{𝑖𝑗𝑘;}

where

Yijk = is the response variable, μ = is the general mean from the experiment, Zni = is the effect of the i-th Zinc level, Aj = the effect of the j-th feeding factor, Zn*Aij = effect of the interaction and Eijk= the random error.

For the intestinal morphology analysis, the mathematical model was the following:

𝑌_𝑖𝑗𝑘 = 𝜇 + 𝑍𝑛_𝑖 + 𝐶_𝑗 + 𝑍𝑛𝐶_𝑖𝑗 + 𝐸_𝑖𝑗𝑘;

where: Yijk = is the response variable, µ = is the general mean of the experiment, Zni = is the effect of the i-th level of Zinc, Cj = the effect of the j-th climate factor, Zn*Cij = effect of the interaction and Eijk = is the random error.

RESULTS AND DISCUSSION

Experiment 1. The results of the effect of the consumption of feed added with Zn methionine (ZnMet) on the productive performance of pigs during the development- completion stage, under conditions of high caloric load, are shown in table 5. In the completion stage (days 50 to 90, of the experimental period), the DWG tended (p = 0.07) to improve. The DFC was higher (p = 0.03) in the pigs of sows supplemented with Zn during GL; observing a trend (P = 0.10) of improvement in total weight gain during this stage. However, when analyzing the entire study period (90 d), supplementation with zinc methionine did not improve the productive performance of pigs.

The results of the effect of the consumption of feed added with ZnMet on the epithelial morphology of the small intestine during the hot and cool season are shown in Tables 7 and 8. The results indicate that the pigs that consumed diets supplemented with 100 mg of Zn/kg of feed, from zinc methionine, during the hot season, had a higher (p <0.01) V:C ratio (3.36 vs 2.77), compared to those that did not consume additional zinc during said period. Consumption of additional zinc decreased (p <0.05) the depth of the crypts in all segments (duodenum, jejunum and ileum) of the small intestine.

Experiment 2. During the cool season of the year the treatments did not modify the productive response of the pig (table 6), nor the morphology of the intestinal epithelium (tables 7 and 8). However, the additional consumption of zinc methionine during the gestation-lactation period tended (p = 0.06) to decrease mortality during the fattening period. In this regard, it has been reported that supplementation with Zn in the sow's diet during gestation and lactation reduces pre-weaning mortality (^{Payne et al., 2006}; ^{Romo et al., 2017}) and improves the immune function of weaned piglets (^{Romo et al., 2017}); which agrees with the results obtained in the present study.

The amount of organic Zn (ZnMet) supplemented to the pigs in the present study was 100 mg/kg of feed, in addition to the inorganic Zn content provided by the diets (120.20 mg/kg in the development diet and 105.31 mg/kg in completion), an amount higher than recommended by the NRC (2012). However, these levels are not toxic and are routinely used in commercial swine diets. The pigs of exp. 2 were exposed to an average temperature of 20.57 °C and relative humidity of 72.0% (table 4; ^{CIAD, 2015}, ²⁰¹⁶); remaining at a THI of 67.39, environmental conditions under which the pig does not suffer physiological stress derived from the physical environment (^{Mader et al., 2006}). While the pigs of experiment 1, they remained in a THI of 81 points (in physiological danger, see table 3) with an average environmental temperature of 29.58 °C and 72.61% relative humidity. The difference in environmental temperature and relative humidity between the pigs in experiment 1, compared to those in experiment 2, was 9.01 °C and 0.61%.

The results of the morphological analysis of the duodenum, jejunum and ileum samples show that the pigs that consumed diets supplemented with 100 mg of Zn/kg of feed, from zinc methionine, during the hot season, had a higher (p <0.01) V: C ratio (3.36 vs 2.77), with respect to those who did not consume additional zinc during said period. Consumption of additional zinc decreased (p <0.05) the depth of the crypts in all segments (duodenum, jejunum and ileum) of the small intestine. An interaction (p <0.02) was observed between the climate and zinc methionine supplementation in the depth of the crypt and in the V:C ratio. The effect of climate and zinc methionine supplementation on the epithelial morphology of each segment of the small intestine analyzed is described below:

Epithelial morphology of the duodenum. The analysis indicates an interaction (p <0.01) of the climate with zinc, observing that in cool climate the pigs have a better response to the additional consumption of 100 mg of Zn/kg of feed, with a greater height of the villi with respect to the pigs supplemented during hot weather. In the case of the depth of the crypts, the pigs that consumed feed added with zinc methionine, both in the warm season and in the cool season, had a lower depth (p <0.02); while the V:C relationship was influenced (p <0.01) by the climate; being older during the cool season.

Jejunum epithelial morphology. The climate was the factor that modified (p <0.01) the height of the villi, observing a higher height of these in the pigs treated during the cool season. However, the depth of the crypts was lower (p <0.05) in the pigs that consumed feed added with zinc methionine, both in the warm and cool seasons, as well as in those that did not consume additional zinc during the cool season. (p <0.05). Similarly, the highest (p <0.03) V: C relationship was d in the animals and times of the year observe previously referred.

Ileum epithelial morphology. In the ileum region, the height of the villi was also higher (p <0.01) in the pigs treated during the cool season. However, the depth of the crypt was lower (p <0.03) in the pigs that consumed feed added with zinc methionine, both in the warm season and in the cool one, observing an interaction (p <0.03) between the factors climate and zinc an interaction (p <0.04) between climate and zinc was found in the V: C ratio.

When doing the pooled analysis (see Table 8) of the epithelial morphology of the three segments of the small intestine, it was observed that the climate has a greater effect (p<0.01) on the height of the villi, and that there is an interaction (p<0.02) between climate and zinc supplementation in reducing crypt depth and increasing V: C ratio.

The results of the present study indicate that supplemental zinc at the level of 100 mg / kg of feed, from zinc methionine, improves the intestinal morphology of fattening pigs reared under a hot environment; but when it is supplemented to pigs raised under cool environments the response is improved.

It has been suggested that a low V: C ratio may indicate villus atrophy, associated with an increased rate of cell loss from the villus apex, concurrent with increased cell production in the crypts, and thus therefore, a greater depth of these. A higher V: C ratio suggests a healthier state of the intestine (^{Tang et al., 1999}). ^{Al et al. (2015)} indicated that the height of the villi and the depth of the crypt show remarkably interdependent developmental changes. Therefore, its morphometric measurement cannot be considered individually; instead the V: C ratio should be evaluated. It suggests that the higher V: C ratio observed in the present study (3.36 vs. 2.77; p <0.01) in pigs that received supplementation with 100 mg of Zn (ZnMet) kg-1 during the hot season, regarding of those who did not receive additional zinc. It is due to the beneficial action of organic Zn on the maintenance of intestinal integrity and morphology. These results are congruent with those obtained by ^{Bouwhuis et al. (2016}), who reported that the inclusion of zinc methionine increased the height of the villi in the jejunum, but did not increase the weight gain of pigs. In addition, studies that are more recent demonstrated that zinc supplementation (450 mg kg-1 of nano-ZnO and 3,000 mg kg-1 of ZnO) protects the morphology of the small intestine, by increasing the height of the villi (^{Long et al., 2017}; ^{Pei et al., 2018}). For their part, ^{Li et al.} (2001) observed that the pigs that received supplemental ZnO had greater thickness of the mucosa and height of the villi, in the proximal and medial sites of the small intestine, with respect to the pigs that received the control diet.

Heat stress is known to affect gastrointestinal health and function (^{Eshel et al., 2001}), since during heat stress blood flow is diverted from the splenic system to the skin, in an attempt to dissipate excess heat (^{Lambert, 2009}). A reduced blood flow and hyperthermia lead to hypoxia, oxidative and nitrosating stress in the enterocyte (^{Pearce et al., 2013}a, b, c), damaging cell and mucous membranes; as well as tight junctions, increasing intestinal permeability (^{Lambert et al., 2002}). In this regard, Pearce et al. (2015) observed that heat stress increases the passage of high molecular weight substances and circulating endotoxins, in addition to an increase in the autolysis of the intestinal epithelium.

Zinc oxide supplementation has been shown to reduce intestinal permeability in piglets at weaning, while increasing the amount and expression of tightly bound proteins (^{Zhang and Guo, 2009}). Also increasing levels of the zinc complex AA (220 and 320 mg/kg) increased the integrity of the gastrointestinal tract, compared to control pigs that received 120 mg/kg of ZnSO4 (^{Sanz-Fernandez et al., 2014}). In addition, this mineral protects cells against oxidative damage by stabilizing membranes, inhibiting the pro-oxidant enzyme nicotinamide adenine dinucleotide phosphate oxidase (NADPH-Oxidase), and inducing metallothionein synthesis (^{Marreiro et al., 2017}). In this regard, it has been reported that zinc supplementation induces the expression of metallothioneins in Caco-2 cells (^{Wang et al., 2013}), which could act as antioxidants, due to their ability to sequester reactive oxygen species and nitrogen intermediates (^{Waeytens et al., 2009}). Furthermore, zinc increases the expression and concentration of antimicrobial substances such as β- defensins in IPEC-J2 cells (^{Mao et al., 2013}).

On the other hand, Zn is a structural component of the enzyme superoxide dismutase (SOD), present in the cytoplasm of cells. SOD promotes the conversion of two superoxide radicals (O2-) into hydrogen peroxide (H2O2) and molecular oxygen (O2), reducing the toxicity of reactive oxygen species (ROS); because it converts a highly reactive species into a less harmful one (^{Cruz and Soares, 2011}; ^{Marreiro et al., 2017}). Studies have also highlighted its role in the regulation of glutathione peroxidase and in the expression of glutamate-cysteine ligase, an enzyme involved in the synthesis of glutathione that acts directly on the neutralization of free radicals (^{Marreiro et al., 2017}). Consequently, there appear to be a variety of mechanisms by which dietary zinc can maintain or restore the integrity and morphology of the intestinal epithelium, under conditions of oxidative stress.

CONCLUSION

Supplementation with 100 mg of zinc/kg of feed, from zinc methionine, improves the integrity of the intestinal epithelium in fattening pigs, reared under conditions of high caloric load, and supplementation during the gestation-lactation period. It can be used as a strategy to reduce mortality during the fattening stage.